Atm kinase compositions and methods

ABSTRACT

The present invention provides methods for detecting activation of ATM kinase, DNA damage, and DNA damaging agents. Further provided are antibodies which specifically recognize the phosphorylation state of Ataxia Telangiectasia-Mutated (ATM) kinase. Methods of identifying agents which modulate the activation and activity of ATM kinase are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 11/285,841,filed Nov. 23, 2005, which is a divisional of application Ser. No.11/076,405, filed Mar. 9, 2005, now U.S. Pat. No. 7,160,692, which is adivisional of application Ser. No. 10/351,733, filed Jan. 24, 2003, nowU.S. Pat. No. 6,916,627, which is a continuation-in-part of U.S.application Ser. No. 10/307,077, filed Nov. 27, 2002, now U.S. Pat. No.7,108,992. These prior applications are incorporated herein by referencein their entirety. This invention was made in the course of researchsponsored by the National Institutes of Health (NIH Grant Nos. CA71387).The U.S. government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

Eukaryotic cells have evolved complex mechanisms to deal withenvironmental stresses. Signal transduction pathways are rapidlyactivated following exposure to DNA damaging agents and other cellularstresses, and these pathways affect processes such as gene transcriptionand cell cycle progression (Hartwell and Weinert (1989) Science246:629-634; Hartwell and Kastan (1994) Science 266:1821-1828; Elledge(1996) Science 274:1664-1672). The protein encoded by theAtaxia-telangiectasia Mutated (ATM) locus, is a kinase critical for theinitiation of signaling pathways following exposure of mammalian cellsto ionizing radiation (IR) and to other agents that introducedouble-strand breaks into cellular DNA (Kastan and Lim (2000) Mol. Cell.Biol. 1:179-186; Shiloh and Kastan (2001) Adv. Cancer Res. 83:209-254).Cells from Ataxia-telangiectasia (A-T) patients typically lackdetectable ATM protein, contain abnormalities in telomere morphology,and exhibit abnormal responses to IR, including increased cell death,increased chromosomal breakage, and cell cycle checkpoint defects(Shiloh (1997) Ann. Rev. Genet. 31:635-662). In addition, A-T patientsexhibit progressive cerebellar ataxia, immune deficiencies, gonadalatrophy, oculocutaneous telangiectasias, radiation sensitivity,premature aging and increased risk of cancers, particularly lymphomas.

The ATM gene encodes a 370-kDa protein (Accession No. Q13315; SEQ IDNO:1) that belongs to the phosphoinositide 3-kinase (PI-3K) superfamily(Savitsky, et al. (1995) Science 268:1749-1753) which phosphorylatesproteins rather than lipids (Banin, et al. (1998) Science 281:1674-1677;Canman, et al. (1998) Science 281:1677-1679). The 350 amino acid kinasedomain at the C-terminus of this protein is the only segment of ATM withan assigned function. Exposure of cells to IR triggers ATM kinaseactivity and this function is required for arrests in G1, S, and G2phases of the cell cycle (Shiloh and Kastan (2001) Adv. Cancer Res.83:209-254). Several substrates of the ATM kinase participate in theseIR-induced cell cycle arrests. For example, phosphorylation of p53,mdm2, and Chk2 govern the G1 checkpoint (Banin, et al. (1998) Science281:1674-1677; Canman, et al. (1998) Science 281:1677-1679; Maya, et al.(2001) Genes Dev. 15:1067-1077; Matsuoka, et al. (2000) Proc. Natl.Acad. Sci. USA 97:10389-10394; Chehab, et al. (2000) Genes Dev.14:278-288); Nbsl, Brcal, FancD2, and SMC1 participate in the transientIR-induced S-phase arrest (Lim, et al. (2000) Nature 404:613-617; Wu, etal. (2000) Nature 405:477-482; Zhou, et al. (2000) J. Biol. Chem.275:10342-10348; Taniguchi, et al. (2002) Cell 109:459-472; Kim, et al.(2002) Genes Dev. 16:560-570; Yazdi, et al. (2002) Genes Dev.16:571-582; Xu, et al. (2002) Cancer Res. 62:4588-4591); and Brcal andhRadl7 have been implicated in the G2/M checkpoint (Xu, et al. (2001)Mol. Cell. Biol. 21:3445-3450; Bao, et al. (2001) Nature 411:969-974).

The mechanisms by which eukaryotic cells sense DNA strand breaks isunknown, but the rapid induction of ATM kinase activity following IRindicates that it acts at an early stage of signal transduction inmammalian cells (Banin, et al. (1998) Science 281:1674-1677; Canman, etal. (1998) Science 281:1677-1679). Transfected ATM is a phosphoproteinthat incorporates more phosphate after IR treatment of cells (Lim, etal. (2000) Nature 404:613-617), suggesting that ATM kinase is itselfactivated by post-translational modification.

Inhibiting ATM for the treatment of neoplasms, particularly cancersassociated with decreased p53 function, has been suggested (Morgan, etal. (1997) Mol. Cell. Biol. 17:2020-2029; Hartwell and Kastan (1994)Science 266:1821-1828; Kastan (1995) New Eng. J. Med. 333:662-663; WO98/56391). WO 98/56391 further provides genetically manipulatedknock-out mice as a model for testing ATM inhibitors and suggests theuse of an inhibitory antibody to ATM, a dominant-negative fragment ofATM or an ATM antisense strategy to inhibit ATM.

U.S. Pat. No. 6,387,640 discloses the use of an ATM kinase substraterecognition sequence in an assay system to screen for compounds thatmodulate ATM-mediated phosphorylation. The substrate recognitionsequence provided comprises Xaa₁-Xaa-Xaa₂-Ser-Gln-Xaa-Xaa (SEQ ID NO:2)wherein Xaa₁ is a hydrophobic amino acid, Xaa₂ is a hydrophobic aminoacid or aspartic acid, and Xaa is any amino acid.

U.S. Pat. No. 6,348,311 further discloses a method of identifying aninhibitor of ATM-mediated kinase activity by determining the extent ofcell survival after HTLV infection.

SUMMARY OF THE INVENTION

One aspect of the present invention provides a method of identifying theactivation state of ATM kinase. In a cell, inactive ATM kinase is foundas a homodimer or higher order multimer. Upon autophosphorylation of aserine corresponding to residue 1981 of ATM kinase (SEQ ID NO:1), ATMbecomes an active monomer. Accordingly, the method provides determiningthe phosphorylation state of serine 1981 (Ser¹⁹⁸¹) which is indicativeof the activation state of ATM kinase. In a preferred embodiment,monoclonal or polyclonal antibodies which specifically recognize thephosphorylation state of Ser¹⁹⁸¹ are used to determine thephosphorylation state of Ser¹⁹⁸¹ of ATM kinase by immunoassay analysis.

Another aspect of the invention provides a method of detecting DNAdamage in a sample comprising identifying the activation state of ATMkinase via the phosphorylation state of Ser¹⁹⁸¹. The method may be usedto monitor the effectiveness of radiation therapy or chemotherapy.

Another aspect of the invention provides a method of detecting DNAdamaging agents in a biological or environmental sample comprisingidentifying the activation state of ATM kinase via the phosphorylationstate of Ser¹⁹⁸¹. A kit for detecting a DNA damaging agent is alsoprovided.

A further aspect of the invention provides a method of producing solubleATM kinase by contacting a first polypeptide of ATM kinase containingthe kinase domain with a second polypeptide of ATM kinase containingSer¹⁹⁸¹. The first and second polypeptides may be produced separately oras a single polypeptide in the same cell.

A further aspect of the invention provides a cell-based assay foridentifying agents which modulate the activation of ATM kinase. Themethod provides contacting cells containing ATM kinase with an agent anddetermining whether said agent agonizes or antagonizes the activation ofATM, kinase in the cell. The activation state of ATM kinase isidentified via the phosphorylation state of Ser¹⁹⁸¹.

A further aspect of the invention provides a cell-free assay foridentifying agents which modulate ATM kinase activity. The methodprovides contacting soluble ATM kinase protein with an agent and ATP anddetermining whether said agent agonizes or antagonizes the ATM kinaseactivity.

These and other aspects of the present invention are set forth in moredetail in the following description of the invention.

DETAILED DESCRIPTION OF THE INVENTION

It has now been found that the major site of ATM kinase phosphorylationis located at serine residue 1981 (Ser¹⁹⁸¹) of ATM kinase (SEQ ID NO:1),also referred to herein as ATM. Phosphorylation of Ser¹⁹⁸¹ results fromintermolecular autophosphorylation. The phosphorylation of Ser¹⁹⁸¹ doesnot directly regulate the activity of the kinase, but instead disruptsATM kinase oligomers which in turn allows accessibility of substrates tothe ATM kinase domain. The rapidity and stoichiometry of thephosphorylation reaction indicate that ATM is not activated by bindingdirectly to DNA strand breaks. While not wishing to be bound by anyparticular theory, it is believed that DNA damage rapidly causes changesin higher order chromatin structures that initiate this activation.

Not to be held to any one particular mechanism of action, it is believedthat in the unperturbed cell, ATM is sequestered as a dimer or higherorder multimer with its kinase domain bound to an internal domain of aneighboring ATM molecule containing Ser¹⁹⁸¹. In this complex, ATM isunable to phosphorylate other cellular substrates. Following DNA damage,the kinase domain of one ATM molecule phosphorylates Ser¹⁹⁸¹ of aninteracting ATM molecule, and the phosphorylated ATM is then dissociatedfrom the complex and is freed to phosphorylate other substrates in thecell. The kinase dead and non-phosphorylatable mutants of ATM retainendogenous ATM in a complex since they cannot be phosphorylated andreleased after IR, thus inhibiting cellular ATM activity. This mechanismprovides an explanation for the dominant inhibitory property ofkinase-inactive ATM.

Several different molecular mechanisms have been identified thatregulate the activity of protein kinases. Protein kinases are generallyrestrained in an inactive state with the acquisition of catalyticactivity controlled at multiple levels, ranging from the binding ofallosteric factors to changes in the subcellular localization of theenzyme (Huse and Kuriyan (2002) Cell 109:275-282). Since all proteinkinases catalyze the same reaction, their active conformations tend tobe structurally similar. However, different classes of kinases haveevolved distinct inactive states and adoption of the catalyticconformation of the enzyme can be impeded in different ways. Theseinclude steric hindrance of substrate access to the catalytic domain byan activation loop that is often controlled by phosphorylation (Johnsonand Noble (1996) Cell 85:149-158); allosteric regulation of theactivation loop via, for example, the PSTAIRE helix in the cyclindependent kinase family (De Bondt, et al. (1993) Nature 363:595-602;pseudosubstrate inhibition of both substrate and nucleotide binding asseen in twitchin (Hu, et al. (1994) Nature 369:581-584); andintramolecular autoinhibition by N-terminal segments that inhibitcatalytic activity, as in the case of the EphB2 receptor kinase(Dodelet, and Pasquale (2000) Oncogene 19:56145619). The resultsprovided herein indicate a novel mechanism of kinase activation in whichthe cellular activity of one ATM kinase molecule is impeded byintermolecular association with an internal domain of a partner ATMmolecule; access of substrates to the catalytic domain is impeded bythis association. This type of regulation is similar to pseudosubstrateinhibition, with the major variations being that the pseudosubstrate isa domain of itself (albeit in trans) and that this partner is not amimic, but actually becomes a substrate in order to release theinhibition.

Accordingly, the present invention provides compositions and methods foridentifying the activation state of ATM kinase by determining thephosphorylation state of Ser¹⁹⁸¹ of ATM kinase. The activation of ATMkinase is indicative of DNA damage; thus, the invention further providesmethods for detecting DNA damage and DNA damaging agents. Furtherprovided is a method for producing soluble ATM kinase by combining afirst polypeptide of ATM kinase containing the kinase domain with asecond polypeptide of ATM kinase containing Ser¹⁹⁸¹. Methods ofidentifying agents which modulate activation of ATM kinase in a cell andATM kinase activity are also provided.

One aspect of the invention provides a method of identifying theactivation state of ATM kinase in a cell. In an unperturbed cell (i.e.,a cell in which no DNA damage has occurred), ATM kinase is found in adimer or multimer which is the inactivate state wherein ATM is unable tophosphorylate other cellular substrates. Upon DNA damage, ATMautophosphorylates Ser¹⁹⁸¹ and is converted into a monomer which is theactive state wherein ATM is able to phosphorylate other cellularsubstrates. Therefore, in a cell, the active and inactive states (i.e.,activation states) of ATM kinase are distinguishable by thephosphorylation state of Ser¹⁹⁸¹ of ATM kinase (SEQ ID NO:1). As one ofskill in the art can appreciate, the activation state of any homolog ormutant of ATM kinase may be identified by determining thephosphorylation state of a serine corresponding to residue 1981 of ATMkinase. The location of the critical serine residue corresponding toSer¹⁹⁸¹ of ATM kinase can readily be determined by comparing thesequence of ATM kinase (SEQ ID NO:1) with the sequence of homologs ormutants of ATM kinase.

In general, the method of identifying the activation state of ATM kinasecomprises obtaining a sample such as a biopsy sample, tissue, cell orfluid (e.g., whole blood or plasma) isolated from a subject anddetermining the phosphorylation state of Ser¹⁹⁸¹ of ATM kinase in thesample. Kinase inhibitors may be present during the isolation of ATMkinase to preserve the phosphorylation state of Ser¹⁹⁸¹ as it would havebeen found in the cell prior to the isolation step. It is contemplatedthat the phosphorylation state of Ser¹⁹⁸¹ of ATM may be determined usinga variety of separation and/or detection methods, including thoseexemplified herein. For example, [³²p] phosphorylated ATM is digestedwith trypsin and separated by well-known conventional columnchromatography, 2-D gel electrophoresis, or capillary electrophoresismethodologies. For separation by column chromatography, reverse-phaseHPLC may be employed with collection via peak detection. Under theconditions used for reverse-phase HPLC (0.05% TFA, pH 2.2), aphosphorylated peptide generally elutes slightly earlier than thecorresponding non-phosphorylated peptide and may or may not be separatedfrom it. Once HPLC fractions containing the Ser¹⁹⁸¹ phosphorylatedpeptide are located by Cerenkov counting, a small aliquot of each may beanalyzed by MALDI-MS.

As an alternative to radiolabeling, western blots made from 2-D gels maybe probed using anti-phosphoserine antibodies (Research Diagnostics,Inc., Flanders, N.J.) to recognize the degree of phosphorylation of apeptide fragment containing Ser¹⁹⁸¹.

Alternatively, one may use a phosphoprotein purification kit (QIAGEN®,Valencia, Calif.) for separation of the phosphorylated from theunphosphorylated cellular protein fraction. The affinity chromatographyprocedure, in which phosphorylated proteins are bound to a column whileunphosphorylated proteins are recovered in the flow-through fraction,reduces complexity and greatly facilitates phosphorylation-profilestudies. ATM may then be purified from each fraction and the degree ofphosphorylation of a peptide fragment containing Ser¹⁹⁸¹ determined viaautoradiography or immunoassays.

In a preferred embodiment, the phosphorylation state of Ser¹⁹⁸¹ of ATMkinase is detected using antibodies which specifically recognize thephosphorylation state of Ser¹⁹⁸¹ of ATM kinase (SEQ ID NO:1). Suchantibodies may be utilized with or without purification, fragmentation,or fractionation or ATM. An antibody which specifically recognizes thephosphorylation state of Ser¹⁹⁸¹ of ATM kinase (SEQ ID NO: 1) comprisespolyclonal antibody α-Ser¹⁹⁸¹, which specifically recognizesunphosphorylated Ser¹⁹⁸¹; and polyclonal antibody α-Ser¹⁹⁸¹-P andmonoclonal antibodies 7C10, 12E10, 13C5, 13H4, 2H12, 7A4, 9D8 and 10H11,which specifically recognize phosphorylated Ser¹⁹⁸¹. An antibody is saidto specifically recognize the phosphorylation state of Ser¹⁹⁸¹ if it isable to discriminate between the unphosphorylated and phosphorylatedforms of Ser¹⁹⁸¹ and bind ATM to form an ATM kinase-antibody complex,i.e., antigen-antibody complex. For example, an antibody whichspecifically recognizes the unphosphorylated state of Ser¹⁹⁸¹ will onlybind to an ATM kinase with an unphosphorylated Ser¹⁹⁸¹ and not to an ATMkinase with a phosphorylated Ser¹⁹⁸¹ (e.g., α-Ser¹⁹⁸¹). Likewise, anantibody which specifically recognizes the phosphorylated state ofSer¹⁹⁸¹ will only bind to an ATM kinase with a phosphorylated Ser¹⁹⁸¹and not to an ATM kinase with an unphosphorylated Ser¹⁹⁸¹ (e.g.,α-Ser¹⁹⁸¹-P, 7C10, 12E10, 13C5, 13H4, 2H12, 7A4, 9D8, 10H11).

In general, a method of using antibodies which specifically recognizethe phosphorylation state of Ser¹⁹⁸¹ in the identification of theactivation state of ATM kinase provides contacting a sample with saidantibody and detecting the formation of an antigen-antibody complexusing an immunoassay. The ATM kinase antigen, as used herein, includesboth the phosphorylated and unphosphorylated states of Ser¹⁹⁸¹ however,the phosphorylated state is preferred. The conditions and time requiredto form the antigen-antibody complex may vary and are dependent on thesample being tested and the method of detection being used. Oncenon-specific interactions are removed by, for example, washing thesample, the antigen-antibody complex is detected using any one of thewell-known immunoassays used to detect and/or quantitate antigens.Exemplary immunoassays which may be used in the methods of the inventioninclude, but are not limited to enzyme-linked immunosorbent,immunodiffusion, chemiluminescent, immunofluorescent,immunohistochemical, radioimmunoassay, agglutination, complementfixation, immunoelectrophoresis, western blots, mass spectrometry,antibody array, and immunoprecipitation assays and the like which may beperformed in vitro, in vivo or in situ. Such standard techniques arewell-known to those of skill in the art (see, e.g., “Methods inImmunodiagnosis”, 2nd Edition, Rose and Bigazzi, eds. John Wiley & Sons,1980; Campbell et al., “Methods and Immunology”, W. A. Benjamin, Inc.,1964; and Oellerich, M. (1984) J. Clin. Chem. Clin. Biochem. 22:895-904;Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, New York (1988) 555-612).

These immunoassays typically rely on labeled antigens, antibodies, orsecondary reagents for detection. These proteins may be labeled withradioactive compounds, enzymes, biotin, or fluorochromes. Of these,radioactive labeling may be used for almost all types of assays.Enzyme-conjugated labels are particularly useful when radioactivity mustbe avoided or when quick results are needed. Biotin-coupled reagentsusually are detected with labeled streptavidin. Streptavidin bindstightly and quickly to biotin and may be labeled with radioisotopes orenzymes. Fluorochromes, although requiring expensive equipment for theiruse, provide a very sensitive method of detection. Those of ordinaryskill in the art will know of other suitable labels which may beemployed in accordance with the present invention. The binding of theselabels to antibodies or fragments thereof may be accomplished usingstandard techniques (see, for example, Kennedy, et al. (1976) Clin.Chim. Acta 70:1-31 and Schurs, et al. (1977) Clin. Chim Acta 81:1-40).

In accordance with identifying the activation state of ATM kinase, thepresence or absence of the antigen-antibody complex is correlated withactive or inactive ATM kinase in a sample, respectively. For example, asample to which, α-Ser¹⁹⁸¹-P binds is indicative of the presence ofactive ATM kinase in said sample.

As provided herein, monoclonal and rabbit polyclonal antibodies thatspecifically recognize Ser¹⁹⁸¹ only when it is in either theunphosphorylated (α-Ser¹⁹⁸¹) or phosphorylated (α-Ser¹⁹⁸¹-P, 7C10,12E10, 13C5, 13H4, 2H12, 7A4, 9D8, 10H11) state were generated. Initialspecificity of these antibodies for appropriate peptides wasdemonstrated by ELISA and by specific recognition and blocking on dotblots. The specificity of the antibodies were confirmed on western blotsof immunoprecipitated FLAG®-tagged ATM protein where wild-type andkinase-inactive ATM were recognized by both antisera, but ATM proteinwith Ser¹⁹⁸¹ mutated to alanine (Ser¹⁹⁸¹→Ala) was not recognized byeither antisera. The relative amount of wild-type ATM recognized by theα-Ser¹⁹⁸¹ antisera was reduced several-fold within 30 minutes afterexposure of the cells to 10 Gy IR, whereas the relative amount ofkinase-inactive ATM that was recognized remained the same. Conversely,the relative amount of wild-type ATM recognized by the α-Ser¹⁹⁸¹-Pantisera was increased several-fold 30 minutes after treatment with 10Gy IR while recognition of kinase-inactive ATM was unaffected. Theseresults mirror the metabolic labeling results using transfectedwild-type ATM and kinase-inactive ATM in 293T cells described above.

Upon exposure to IR and UV, endogenous ATM is phosphorylated at Ser¹⁹⁸¹.Non-transformed, exponentially growing primary human fibroblasts wereexposed to either 10 Gy IR or 10 J/m² UV. The α-Ser¹⁹⁸¹-P antisera didnot bind to ATM protein immunoprecipitated from unirradiated cells, butrecognized ATM one hour following exposure to IR and five hoursfollowing exposure to both IR and UV. The relative amount of Ser¹⁹⁸¹phosphorylation seen five hours following IR treatment was several-foldhigher than that seen following UV. This differential recognition wasnot due to changes in cell cycle distribution which did not changesignificantly in the first hour after IR. Moreover, primary fibroblastsarrested in G₀ also demonstrate this phosphorylation event followingexposure to either IR or UV irradiation.

Antibodies provided in the present disclosure are of the monoclonal andpolyclonal type. It is contemplated that such antibodies may be naturalor partially or wholly synthetically produced. All fragments orderivatives thereof which maintain the ability to specifically bind toand recognize the phosphorylation state of Ser¹⁹⁸¹ of ATM kinase arealso contemplated. The antibodies may be a member of any immunoglobulinclass, including any of the classes: IgG, IgM, IgA, IgD, and IgE.Derivatives of the IgG class, however, are preferred in the presentinvention.

ATM kinase antibody fragments may be any derivative of an antibody whichis less than full-length. Preferably, the antibody fragment retains atleast a significant portion of the full-length antibody's specificbinding ability. Examples of antibody fragments include, but are notlimited to, Fab, Fab′, F(ab′)2, scFv, Fv, dsFv diabody, or Fd fragments.The antibody fragment may be produced by any means. For instance, theantibody fragment may be enzymatically or chemically produced byfragmentation of an intact antibody or it may be recombinantly producedfrom a gene encoding the partial antibody sequence. The antibodyfragment may optionally be a single-chain antibody fragment.Alternatively, the fragment may comprise multiple chains which arelinked together, for instance, by disulfide linkages. The fragment mayalso optionally be a multi-molecular complex. A functional antibodyfragment will typically comprise at least about 50 amino acids and moretypically will comprise at least about 200 amino acids. As used herein,an antibody also includes bispecific and chimeric antibodies.

Naturally produced antibodies may be generated using well-known methods(see, e.g., Kohler and Milstein (1975) Nature 256:495-497; Harlow andLane, In: Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, New York (1988)). Alternatively, ATM kinase antibodies whichspecifically recognize the phosphorylation state of Ser¹⁹⁸¹ of ATMkinase are derived by a phage display method. Methods of producing phagedisplay antibodies are well-known in the art (e.g., Huse, et al. (1989)Science 246(4935):1275-81).

Selection of ATM kinase-specific antibodies is based on binding affinityto ATM kinase which is either phosphorylated or unphosphorylated atSer¹⁹⁸¹ and may be determined by the various well-known immunoassaysindicated above.

Another aspect of the present invention provides a method of detectingDNA damage in a cell by identifying the activation state of ATM kinase.It has now been shown that DNA damaging agents which introduce breaks inthe phosphodiester backbone of DNA induce rapid, detectablephosphorylation of Ser¹⁹⁸¹ and hence activation of ATM kinase. Ser¹⁹⁸¹phosphorylation was examined in exponentially growing primaryfibroblasts one and five hours after treatment with IR, thymidine block,the ribonucleotide reductase inhibitor hydroxyurea (HU), thetopoisomerase inhibitors camptothecin and etoposide, the DNA alkylatingagent methlymethanesulfonate (MMS), the DNA polymerase α inhibitoraphidicolin, or the oxidizing agent H₂O₂. Phosphorylation of Ser¹⁹⁸¹ inATM was detected one hour following exposure to 10 Gy IR, 2 nMcamptothecin, 17 nM etoposide or 0.1% H₂O₂, all of which are DNAdamaging agents. The only significant change in this pattern five hoursafter each of these treatments was more prominent phosphorylationfollowing exposure to the alkylating agent, MMS. The delayed Ser¹⁹⁸¹phosphorylation after MMS was similar to that observed following UVirradiation, an agent which, like MMS, damages DNA bases, but can induceDNA strand breaks either through repair processes or by DNA replicationpast DNA adducts. Little or no phosphorylation of ATM was apparent overthis time frame following exposure to the DNA synthesis inhibitorshydroxyurea, thymidine, or aphidicolin, none of which directly damageDNA. Thus, all agents tested that directly damage DNA by, for example,inducing DNA strand breaks, induced rapid phosphorylation of Ser¹⁹⁸¹,whereas treatments that primarily inhibit DNA synthesis failed to do so.

The kinetics, dose responsiveness and stoichiometry of ATMautophosphorylation following IR were also examined in primaryfibroblasts. Over a 24-hour time frame, phosphorylation of Ser¹⁹⁸¹ wasmaximal by 15 minutes after exposure to 2 Gy IR and remained stable anddetectable for at least 24 hours thereafter. Moreover, phosphorylationof Ser¹⁹⁸¹ was detected immediately upon cellular harvesting followingthe 30 second exposure required to deliver 0.5 Gy IR and was maximal byfive minutes. Initial dose responsiveness was evaluated over a rangefrom 1 to 9 Gy, but induction was already maximal at 1 Gy at the 30minute time point used. A more detailed evaluation of doses less than 1Gy provided that phosphorylation of Ser¹⁹⁸¹ was detectable following0.11 Gy and was already maximal following 0.44 Gy at a 15 minute timepoint.

As the phosphorylation of Ser¹⁹⁸¹ in ATM can be detected followingexposure to doses of IR as low as 0.11 Gy, which theoretically shouldcause just four double-strand breaks in the genomic DNA of a humandiploid cell (Rogakou, et al., (1998) J. Biol. Chem. 273:5858-5868).,the minimal number of DNA double-strand breaks that would inducedetectable ATM phosphorylation was determined. SV-40-transformedfibroblasts were obtained that had been stably transfected with aplasmid containing a sequence that can be cut by the restriction enzymeI-SceI, a site which has not been found in any mammalian genome(Richardson, et al. (1999) Methods Mol. Biol. 113:453-463). Southernblotting demonstrated that the genome of this cell line contained twocopies of the I-SceI site. The α-Ser¹⁹⁸¹-P antibody was able to detectSer¹⁹⁸¹ phosphorylation of FLAG®-tagged, wild-type ATM that wasco-transfected with I-SceI. No phosphorylation was seen in controltransfectants where no active I-SceI was introduced or when eitherkinase-inactive ATM or ATM mutated at Ser¹⁹⁸¹ was utilized. Therefore,the α-Ser¹⁹⁸¹-P antibody can detect the introduction of as few as twoDNA double-strand breaks in cells.

To estimate the fraction of cellular ATM protein that becomesphosphorylated after DNA damage, sequential immunoprecipitations of ATMfrom irradiated primary fibroblasts were performed with a conventionalanti-ATM antibody and with the α-Ser¹⁹⁸¹-P antisera. In the absence ofinsult, the conventional anti-ATM antibody was able to immunoprecipitatevirtually all of the ATM in the first absorption from unirradiatedcells, while the α-Ser¹⁹⁸¹-P antisera brought down almost no ATM. Thelittle ATM that was immunoprecipitated by the α-Ser¹⁹⁸¹-P antisera wasnot recognized by this antibody on western blots, likely due to a verysmall amount of non-phospho-specific antisera in the polyclonal antibodypreparation. Following exposure to 0.5 Gy IR, the amount of ATMimmunoprecipitated by the α-Ser¹⁹⁸¹-P antisera in the first absorptionwas similar to the amount of ATM immunoprecipitated by the conventionalanti-ATM antibody and was greater than the remaining cellular ATM thatwas brought down in the second absorption by the conventional anti-ATMantibody. Since the conventional anti-ATM antibody was not completelyefficient in bringing down all of the cellular ATM with a singleimmunoprecipitation from the irradiated cells it was estimated that atleast 50% of the total ATM in an exponentially growing culture ofprimary human fibroblasts is autophosphorylated on Ser¹⁹⁸¹ by 15 minutesafter exposure to 0.5 Gy IR (estimated 18 DNA breaks).

Since such a high fraction of cellular ATM becomes phosphorylated sorapidly in the presence of so few DNA strand breaks, it is unlikely thatthe ATM oligomers could require direct binding to DNA strand breaks foractivation and autophosphorylation. Therefore, the introduction of DNAstrand breaks may cause a change in the nucleus that activates ATM at adistance from the break itself. As DNA strand breaks introduced byionizing irradiation rapidly alter topological constraints on DNA (RotiRoti and Wright (1987) Cytometry 8:461-467; Jaberaboansari, et al.(1988) Radiat. Res. 114:94-104; Malyapa, et al. (1996) Int. J. Radiat.Oncol. Biol. Phys. 35:963-973), alterations in some aspect of chromatinstructure fit the criteria of being a rapid change and being able to acta distance in the nucleus. Chromatin and chromosome structures can bealtered in the absence of DNA breaks by hypotonic conditions (Earnshaw,and Laemmli (1983) J. Cell Biol. 96:84-93; Jeppesen, et al. (1992)Chromosoma 101:322-332), exposure to chloroquine (Krajewski (1995) FEBSLetters 361:149-152; Jensen, et al. (1994) Cancer Res. 54:2959-2963;Snyder (2000) Environ. Mol. Mutagen. 35:13-21), or treatment withhistone deaceylase inhibitors (Krajewski (1999) J. Biomol. Struct.Dynam. 16:1097-1106; Yoshida, et al. (1995) BioEssays 17:423-430; Kuoand Allis (1998) BioEssays 20:615-626). Exposure of cells to mildlyhypotonic buffers, chloroquine or Trichostatin induced rapid and diffusephosphorylation of ATM protein as assessed by immunoblot andimmunofluorescence. No phosphorylation of histone H2AXγ was observed,therefore there was no evidence for the introduction of DNA strandbreaks with these treatments. Interestingly, these chromatin-modifyingtreatments induced phosphorylation of p53, whose phosphorylation by ATMdoes not occur at the site of DNA breaks. In contrast, phosphorylationof both ATM and H2AXγ were apparent following IR, whether it was usedalone or in combination with other treatments. Additionally, all threeof these agents were able to enhance the amount of ATM phosphorylationseen after exposure of cells to sub-maximal (0.2 Gy) doses of IR. Thepatterns of immunofluorescent staining for ATM following that at theearliest time points, the staining was diffuse across the nucleus, butafter several minutes, some foci were seen in addition to the diffusenuclear staining. This pattern is consistent with a diffuse activationof ATM and migration of a fraction of ATM protein to the sites of DNAstrand breaks to phosphorylate substrates at the breaks. Diffuseimmunofluorescence, but no ATM foci, was seen when staining the cellswith the α-Ser¹⁹⁸¹ antibody.

Accordingly, in another preferred embodiment DNA damage in a sample isdetected by identifying the activation state of ATM kinase. The methodprovides obtaining a sample from a subject and determining thephosphorylation state of Ser¹⁹⁸¹ of ATM kinase. The phosphorylationstate of Ser¹⁹⁸¹ of ATM kinase may be determined using any one of thetechniques provided herein, however, it is preferred that antibodieswhich specifically recognize the phosphorylation state of Ser¹⁹⁸¹ of ATMkinase be used. A sample containing a phosphorylated Ser¹⁹⁸¹ of ATMkinase, as determined by, for example, the binding of α-Ser¹⁹⁸¹-Pantibody, is indicative of active ATM kinase and hence DNA damage in thesubject from which the sample was obtained. Accordingly, a method ofdetecting DNA damage may be used as part of a screen in subjectssuspected of having been exposed to a DNA damaging agent. Moreover, thedetection method of the invention may be used alone or in combinationwith other well-known diagnostic methods to confirm DNA damage.

Damage to DNA may have a genetic- or age-related basis or may resultfrom exposure to agents including those which generate DNA adducts byalkylation (e.g., methylmethane sulfonate (MMS), ethylmethane sulfonate(EMS), N-methyl-N-nitro-N-nitrosoguanine (MNNG), dimethylnitrosamine(DMN), dimethyl sulfate), and form intra- and inter-strand crosslinks(e.g., mitomycin C, psoralens). Furthermore, exposure to base analogs,such as bromouracil and aminopurine; nitrous acid; large molecules whichbind to bases in DNA and cause them to be noncoding, i.e., “bulky”lesions; chemicals causing DNA strand breaks (e.g., peroxides); andradiation such as ultraviolet and ionizing radiation (e.g., X- andgamma-rays) also result in DNA damage.

Detection of DNA damage in a cell as determined by the activation stateof ATM kinase is also useful for monitoring therapeutic effects duringclinical trials and other treatment. Thus, the therapeutic effectivenessof an agent, such as a radionucleide for radiation therapy or acytotoxic agent for chemotherapy designed to cause DNA damage in a cell,can be monitored using the phosphorylation state of Ser¹⁹⁸¹ of ATMkinase, i.e., active ATM kinase, as an end-point target.

A further aspect of the invention provides a method of detecting a DNAdamaging agent in a sample. A sample may be either of biological orenvironmental origin. Biological samples include those provided above aswell as food products and ingredients such as dairy items, vegetables,meat and meat by-products, and waste. Environmental samples includeenvironmental material such as surface matter, soil, water, wastewater,sewage, sludge, industrial samples (e.g., industrial water), as well assamples obtained from food and dairy processing instruments, apparatus,equipment, disposable and non-disposable items. In addition to theseenvironmental samples, it is contemplated that drinking water may beused with the method of the present invention. It is intended that theterm drinking water encompass all types of water used for consumption byhumans and other animals, including but not limited to well water,run-off water, water stored in reservoirs, rivers, streams, etc. Themethod provides contacting a test cell, which can be of prokaryotic oreukaryotic origin, containing ATM kinase, with a sample suspected ofhaving a DNA damaging agent, allowing the test cell to incubate in thepresence of the sample, and detecting whether DNA damage has occurred inthe test cell by identifying the activation state of ATM kinase in saidcell. Methods for identifying the activation state of ATM kinase in acell are provided herein. In a preferred embodiment, active ATM kinase,in a cell exposed to a DNA damaging agent, is identified by determiningthe phosphorylation state of Ser¹⁹⁸¹ using antibodies which specificallyrecognize the phosphorylation state of Ser¹⁹⁸¹ of ATM kinase.

A further aspect of the invention provides a kit to detect a DNAdamaging agent in a sample. The kit comprises antibodies whichspecifically recognize the phosphorylation state of Ser¹⁹⁸¹ of ATMkinase and are preferably labeled. The kit may further comprise a testcell containing ATM kinase. Further provided in the kit may be a meansfor determining the antigen-antibody complex using, for example, animmunoassay, and means for comparing the amount of antigen-antibodycomplex with a standard. The kit may be packaged in a suitable containerand further comprise instructions for using the kit to detect DNAdamaging agents.

A further aspect of the invention provides a method of producing solubleATM kinase by contacting the kinase domain of ATM with a fragment of ATMkinase containing Ser¹⁹⁸¹. The domain of ATM containing Ser¹⁹⁸¹ wasfound to stably interact with the kinase domain of ATM. A GST fusionprotein containing residues 1961-2046 of ATM (GST-ATM¹⁹⁶¹⁻²⁰⁴⁶) wasco-expressed in E. coli with a 6×-Histidine (6×-His) fusion proteincontaining the C-terminal kinase domain of ATM, residues 2712-3056.Although the kinase domain was insoluble in bacteria when co-transfectedwith GST alone or a GST fusion with the ATM target peptide p53 (residues1-101), a significant percentage was stabilized and solubilized in thepresence of the GST-ATM¹⁹⁶¹⁻²⁰⁴⁶ fusion protein. The kinase domain wasalso solubilized in the presence of the GST-ATM¹⁹⁶¹⁻²⁰⁴⁶ fusion proteincontaining Ser¹⁹⁸¹→Ala, but remained insoluble in the presence ofphosphorylation-mimic fusion peptides, Ser¹⁹⁸¹→Asp and Ser¹⁹⁸¹→Glu.Furthermore, the soluble kinase domain co-purified onglutathione-agarose with the GST-ATM¹⁹⁶¹⁻²⁰⁴⁶ fusion proteins containingwild-type sequence or Ser¹⁹⁸¹→Ala. These results indicate that thekinase domain and phosphorylation domain can stably bind to one anotherand that sequences flanking Ser¹⁹⁸¹ are critical for this interaction.The interaction was prevented by mutation of Ser¹⁹⁸¹ to either asparticacid (Asp) or glutamic acid (Glu), both of which have charged sidechains that mimic serine phosphorylation, indicating thatphosphorylation of Ser¹⁹⁸¹ may prevent interaction of this domain withthe kinase domain.

Diploid human fibroblasts were exposed to a range of formaldehydeconcentrations to covalently crosslink endogenous ATM into a minimalcomplex. A prominent complex containing ATM was immunoprecipitated fromcells following treatment of cells with 0.5 mM or 1.0 mM formaldehydefor 10 minutes. This complex migrated electrophoretically considerablyslower than the denatured ATM monomer, which runs at 370-kDa, and beyondthe range of conventional molecular weight markers. Furthermore, thisATM-containing complex disappeared after exposure of the cells to 10 GyIR and was not recognized by the α-Ser¹⁹⁸¹-P antibody.

The number of ATM molecules present in the complex as well as thedependence of Ser¹⁹⁸¹ phosphorylation on the dissociation of the complexwas determined. Hemagglutinin-tagged (HA-tagged) ATM was transfectedinto 293T cells along with wild-type, kinase-inactive, or Ser¹⁹⁸¹→AlaFLAG®-tagged ATM. In the absence of formaldehyde crosslinking, HA-taggedATM was immunoprecipitated by anti-FLAG® Sepharose in association witheach of the three FLAG®-tagged ATM proteins. Following 2 Gy IR,HA-tagged ATM was immunoprecipitated with both kinase-inactive andSer¹⁹⁸¹→Ala FLAG®-tagged ATM, but was no longer bound to wild-typeFLAG®-tagged ATM. In the absence of irradiation, the relative amounts ofkinase-inactive and Ser¹⁹⁸¹→Ala FLAG®-tagged ATM thatco-immunoprecipitated with HA-tagged ATM were lower than that ofwild-type FLAG®-tagged ATM. This indicated that the association ofwild-type, FLAG®-tagged ATM with wild-type, HA-tagged ATM was morestable than that of the mutant, FLAG®-tagged ATMs with HA-tagged ATM.Therefore, ATM exists as a dimer or higher order multimer in unperturbedcells and that intermolecular ATM autophosphorylation on Ser¹⁹⁸¹ isrequired for the dissociation of the complex following DNA damage.

As the ATM kinase domain interacts stably with the domain containing theautophosphorylation site, it was determined. whether a truncatedrecombinant ATM molecule that included the phosphorylation and kinasedomains would fold properly and have kinase activity. Agalactose-inducible pYES plasmid containing 6×-His-FLAG®-taggedATM¹⁹²³⁻³⁰⁵⁶ was transfected into Saccharomyces cerevisiae and a solubleATM fragment was recovered. The polypeptide was purified by anti-FLAG®affinity chromatography. Following elution with synthetic FLAG® peptideand subsequent size-exclusion chromatography, the highly purified ATMfragment had kinase activity, exhibited wortmannin-sensitivephosphorylation of a p53 target peptide and autophosphorylation ofSer¹⁹⁸¹. Some of this ATM fragment migrated at the size of a dimer evenunder the harsh denaturing conditions of the SDS-PAGE gel indicatingthat the ATM protein homodimerizes in cells.

Accordingly, in another embodiment of the invention, soluble ATM kinaseis produced by contacting a first polypeptide comprising the kinasedomain of ATM with a second polypeptide comprising a fragment of ATMkinase containing Ser¹⁹⁸¹. The first and second polypeptide may beproduced separately or produced as a single polypeptide. When producedseparately, the first polypeptide containing the kinase domain maycomprise the entire ATM kinase (i.e., full-length ATM kinase; residues1-3056 of SEQ ID NO:1) or a kinase domain fragment which retains theactivity of ATM kinase (e.g., residues 2712-3056 of SEQ ID NO:1).Furthermore, the second polypeptide containing Ser¹⁹⁸¹ may comprise apolypeptide of 50, 75, 85, or 95 amino acid residues with Ser¹⁹⁸¹located approximately in the center of the polypeptide. An exemplarysecond polypeptide. comprises residues 1961-2046 of SEQ ID NO:1. Whenproduced as a single polypeptide, the nucleic acid sequences encodingthe first and second polypeptide reside on a single contiguous nucleicacid molecule, i.e., translated from one messenger RNA. A singlepolypeptide may comprise, for example, residues 1923-3056 or 1961-3056of SEQ ID NO:1. Alternatively, the single polypeptide may have the firstand second polypeptide separated by a linker peptide ranging from 1 to1000 amino acid residues (e.g., Gly-Ser-Gly, (Gly)₃, and the like).

When produced separately, the first and second polypeptide may beexpressed from the same or separate expression vectors, from the same ordifferent promoters, in the same or separate cells and combined forpurification. Preferably the first and second polypeptide are expressedin the same cell.

Whether produced as a single polypeptide or as separate first and secondpolypeptides, the soluble ATM kinase will be referred to hereinafter asthe ATM kinase polypeptide or simply ATM kinase in the context ofproducing soluble-ATM kinase. Methods of producing ATM kinase in vivo(i.e., cell-based) are provided, however, as will be appreciated by oneof skill in the art, ATM kinase may be produced using well-known invitro transcription and translation methods.

Nucleic acids encoding ATM kinase may be incorporated into a recombinantexpression vector in a form suitable for expression of the proteins in ahost cell. A suitable form for expression provides that the recombinantexpression vector includes one or more regulatory sequencesoperatively-linked to the nucleic acids encoding ATM kinase in a mannerwhich allows for transcription of the nucleic acids into mRNA andtranslation of the mRNA into the protein. Regulatory sequences mayinclude promoters, enhancers and other expression control elements(e.g., polyadenylation signals). Such regulatory sequences are known tothose skilled in the art and are described in Goeddel, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). It should be understood that the design of the expression vectormay depend on such factors as the choice of the host cell to betransfected and/or the level of expression required.

The soluble ATM kinase of the invention may be expressed not onlydirectly, but also as a fusion protein with a heterologous polypeptide,i.e. a signal sequence for secretion and/or other polypeptide which willaid in the purification of ATM kinase. Preferably, the heterologouspolypeptide has a specific cleavage site to remove the heterologouspolypeptide from ATM kinase.

In general, a signal sequence may be a component of the vector andshould be one that is recognized and processed (i.e., cleaved by asignal peptidase) by the host cell. For production in a prokaryote, aprokaryotic signal sequence from, for example, alkaline phosphatase,penicillinase, lpp, or heat-stable enterotoxin II leaders may be used.For yeast secretion, one may use, e.g., the yeast invertase, alphafactor, or acid phosphatase leaders, the Candida albicans glucoamylaseleader (EP 362,179), or the like (see, for example WO 90/13646). Inmammalian cell expression, signal sequences from secreted polypeptidesof the same or related species, as well as viral secretory leaders, forexample, the herpes simplex glycoprotein D signal may be used.

Other useful heterologous polypeptides which may be fused to ATM kinaseinclude those which increase expression or solubility of the fusionprotein or aid in the purification of the fusion protein by acting as aligand in affinity purification. Typical fusion expression vectorsinclude those exemplified herein as well as pGEX (Amersham PharmaciaBiotech, Uppsala, Sweden; Smith, and Johnson (1988) Gene 67:31-40), pMAL(New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway,N.J.) which fuse GST, maltose E binding protein, or protein A,respectively, to the target recombinant protein.

Eukaryotic microbes such as yeast may be transformed with suitablevectors containing nucleic acids encoding ATM kinase. Saccharomycescerevisiae is the most commonly studied lower eukaryotic hostmicroorganism, although a number of other species are commonlyavailable. Yeast vectors may contain an origin of replication from the 2micron yeast plasmid or an autonomously replicating sequence (ARS), apromoter, nucleic acid sequences encoding ATM kinase, sequences forpolyadenylation and transcription termination, and nucleic acidsequences encoding a selectable marker. Exemplary plasmids include YRp7(Stinchcomb, et al. (1979) Nature 282:39; Kingsman, et al. (1979) Gene7:141; Tschemper, et al. (1980) Gene 10:157), pYepSec1 (Baldari, et al.(1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell30:933-943), pJRY88 (Schultz, et al. (1987) Gene 54:113-123), and pYES2(INVITROGEN™ Corporation, San Diego, Calif.). These plasmids containgenes such as trp1, which provides a selectable marker for a mutantstrain of yeast lacking the ability to grow in the presence oftryptophan, for example ATCC No. 44076 or PEP4-1 (Jones (1977) Genetics85:12). The presence of the trp1 lesion in the yeast host cell genomethen provides an effective environment for detecting transformation bygrowth in the absence of tryptophan.

Suitable sequences for promoting ATM kinase expression in yeast vectorsinclude the promoters for metallothionein, 3-phosphoglycerate kinase(Hitzeman, et al. (1980) J. Biol. Chem. 255:2073) or other glycolyticenzymes (Hess, et al. (1968) J. Adv. Enzyme Reg. 7:149; Holland, et al.(1978) Biochemistry 17:4900), such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Suitable vectors andpromoters for use in yeast expression are further disclosed in EP73,657.

In plant cells, expression systems are often derived from recombinant Tiand Ri plasmid vector systems. In the cointegrate class of shuttlevectors, the gene of interest is inserted by genetic recombination intoa non-oncogenic Ti plasmid that contains both the cis-acting andtrans-acting elements required for plant transformation. Exemplaryvectors include the pMLJ1 shuttle vector (DeBlock, et al. (1984) EMBO J.3:1681-1689) and the non-oncogenic Ti plasmid pGV2850 (Zambryski, et al.(1983) EMBO J. 2:2143-2150). In the binary system, the gene of interestis inserted into a shuttle vector containing the cis-acting elementsrequired for plant transformation. The other necessary functions areprovided in trans by the non-oncogenic Ti plasmid. Exemplary vectorsinclude the pBIN19 shuttle vector (Bevan (1984) Nucl. Acids Res.12:8711-8721) and the non-oncogenic Ti plasmid pAL4404 (Hoekema, et al.(1983) Nature 303:179-180).

Promoters used in plant expression systems are typically derived fromthe genome of plant cells (e.g., heat shock promoters; the promoter forthe small subunit of RUBISCO; the promoter for the chlorophyll a/bbinding protein) or from plant viruses (e.g., the 35S RNA promoter ofCaMV; the coat protein promoter of TMV).

In mammalian cells the recombinant expression vector may be a plasmid.Alternatively, a recombinant expression vector may be a virus, or aportion thereof, which allows for expression of a nucleic acidintroduced into the viral nucleic acid. For example,replication-defective retroviruses, adenoviruses and adeno-associatedviruses may be used. Protocols for producing recombinant retrovirusesand for infecting cells in vitro or in vivo with such viruses may befound in Current Protocols in Molecular Biology, Ausubel, F. M. et al.(eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 andother standard laboratory manuals. Examples of suitable retrovirusesinclude, but are not limited to, pLJ, pZIP, pWE and pEM which arewell-known to those skilled in the art. Examples of suitable packagingvirus lines include, but are not limited to, ψCrip, ψCre, ψ2 and ψA1.The genome of adenovirus may be manipulated such that it encodes andexpresses ATM kinase but is inactivated in terms of its ability toreplicate in a normal lytic viral life cycle (Berkner, et al. (1988)BioTechniques 6:616; Rosenfeld, et al. (1991) Science 252:431-434;Rosenfeld, et al. (1992) Cell 68:143-155). Suitable adenoviral vectorsderived from the adenovirus strain Ad type 5 d1324 or other strains ofadenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well-known to those skilled inthe art. Alternatively, an adeno-associated virus vector such as thattaught by Tratschin, et al. ((1985) Mot. Cell. Biol. 5:3251-3260) may beused to express ATM kinase.

In mammalian expression systems, the regulatory sequences are oftenprovided by the viral genome. Commonly used promoters are derived frompolyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For example,the human cytomegalovirus IE promoter (Boshart, et al. (1985) Cell41:521-530), HSV-Tk promoter (McKnight, et al. (1984) Cell 37:253-262)and β-actin promoter (Ng, et al. (1985) Mol. Cell. Biol. 5:2720-2732)may be useful in the expression of ATM kinase in mammalian cells.Alternatively, the regulatory sequences of the recombinant expressionvector may direct expression of ATM kinase preferentially in aparticular cell type, i.e., tissue-specific regulatory elements can beused. Examples of tissue-specific promoters which may be used include,but are not limited to, the albumin promoter (liver-specific; Pinkert,et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calameand Eaton (1988) Adv. Immunol. 43:235-275), promoters of T cellreceptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji, et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund, et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316; EP 264,166).Developmentally-regulated promoters are also encompassed, for examplethe murine hox promoters (Kessel and Gruss (1990) Science 249:374-379)and the α-fetoprotein promoter (Camper and Tilghman (1989) Genes Dev.3:537-546).

When the host cell is from an insect (e.g., Spodoptera frugiperdacells), expression vectors such as the baculovirus expression vector(e.g., vectors derived from Autographa californica MNPV, Trichoplusia niMNPV, Rachiplusia ou MNPV; or Galleria ou MNPV, as described in U.S.Pat. Nos. 4,745,051 and 4,879,236) may be employed to express ATMkinase. In general, a baculovirus expression vector comprises abaculovirus genome containing nucleic acid sequences encoding ATM kinaseinserted into the polyhedrin gene at a position ranging from thepolyhedrin transcriptional start signal to the ATG start site and underthe transcriptional control of a baculovirus polyhedrin promoter.

Escherichia coli is the most common prokaryotic expression system.Exemplary E. coli strains include W3110 (ATCC 27325), E. coli B, E. coliX1776 (ATCC 31537), and E. coli 294 (ATCC 31446). E. coli is typicallytransformed using pBR322 (Bolivar, et al. (1977) Gene 2:95) andderivatives thereof.

Promoters most commonly used in recombinant prokaryotic expressionvectors include the beta-lactamase (penicillinase) and lactose promotersystems (Chang, et al. (1978) Nature 275:615; Goeddel, et al. (1979)Nature 281:544), a tryptophan (trp) promoter system (Goeddel, et al.(1980) Nucl. Acids Res. 8:4057; EP 36,776) the tac promoter (De Boer, etal. (1983) Proc. Natl. Acad. Sci. USA 80:21) and pL of bacteriophage 1.These promoters and Shine-Dalgarno sequence may be used for efficientexpression of ATM kinase in prokaryotes.

ATM kinase is expressed in a cell by introducing nucleic acid sequencesencoding ATM kinase into a host cell, wherein the nucleic acids are in aform suitable for expression of ATM kinase in the host cell.Alternatively, nucleic acid sequences encoding ATM kinase which areoperatively-linked to regulatory sequences (e.g., promoter sequences)but without additional vector sequences may be introduced into a hostcell. As used herein, a host cell is intended to include any prokaryoticor eukaryotic cell or cell line so long as the cell or cell line is notincompatible with the protein to be expressed, the selection systemchosen or the fermentation system employed. Exemplary examples ofmammalian cell lines include, but are not limited to, those exemplifiedherein as well as CHO dhfr-cells (Urlaub and Chasin (1980) Proc. Natl.Acad. Sci. USA 77:4216-4220), 293 cells (Graham, et al. (1977) J. Gen.Virol. 36:59) or myeloma cells like SP2 or NSO (Galfre and Milstein(1981) Meth. Enzymol. 73(B):3-46).

Soluble ATM kinase may be produced in by a variety of non-mammalianeukaryotic cells as well, including insect (e.g., Spodopterafrugiperda), yeast (e.g., S. cerevisiae, Schizosaccharomyces pombe,Pichia pastoris, Kluveromyces lactis, Hansenula Polymorpha and Candidaalbicans, and fungal cells (Neurospora crassa, Aspergillus nidulins,Aspergillus fumigatus).

Nucleic acid sequences encoding ATM kinase may be introduced into a hostcell by standard techniques for transforming cells. Transformation ortransfection are intended to encompass all conventional techniques forintroducing nucleic acid into host cells, including calcium phosphateco-precipitation, DEAE-dextran-mediated transfection, lipofection,electroporation, microinjection, polyethylene glycol-mediatedtransformation, viral infection, Agrobacterium-mediated transformation,cell fusion, and ballistic bombardment. Suitable methods fortransforming host cells may be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratorypress (1989)) and other laboratory manuals.

The number of host cells transformed with a nucleic acid sequenceencoding ATM kinase will depend, at least in part, upon the type ofrecombinant expression vector used and the type of transformationtechnique used. Nucleic acids may be introduced into a host celltransiently, or more typically, for long-term expression of ATM kinase,the nucleic acid is stably integrated into the genome of the host cellor remains as a stable episome in the host cell. Plasmid vectorsintroduced into mammalian cells are typically integrated into host cellDNA at only a low frequency. In order to identify these integrants, agene that contains a selectable marker (e.g., drug resistance) isgenerally introduced into the host cells along with the nucleic acids ofinterest. Preferred selectable markers include those which conferresistance to certain drugs, such as G418 and hygromycin. Selectablemarkers may be introduced on a separate plasmid from the nucleic acidsof interest or introduced on the same plasmid. Host cells transfectedwith nucleic acid sequences encoding ATM kinase (e.g., a recombinantexpression vector) and a gene for a selectable marker may be identifiedby selecting for cells using the selectable marker. For example, if theselectable marker encodes a gene conferring. neomycin resistance, hostcells which have taken up nucleic acid may be selected with G418resistance. Cells that have incorporated the selectable marker gene willsurvive, while the other cells die.

A host cell transformed with nucleic acid sequences encoding ATM kinasemay be further transformed with one or more nucleic acids which serve asthe target for ATM kinase.

Nucleic acid sequences encoding ATM kinase may be introduced into cellsgrowing in culture in vitro by conventional transformation techniques(e.g., calcium phosphate precipitation, DEAE-dextran transfection,electroporation, etc.). Nucleic acids may also be transferred into cellsin vivo, for example by application of a delivery mechanism suitable forintroduction of nucleic acid into cells in vivo, such as retroviralvectors (see e.g., Ferry, et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Kay, et al. (1992) Hum. Gene Ther. 3:641-647), adenoviralvectors (see e.g., Rosenfeld (1992) Cell 68:143-155; Herz and Gerard(1993) Proc. Natl. Acad. Sci. USA 90:2812-2816), receptor-mediated DNAuptake (see e.g., Wu and Wu (1988) J. Biol. Chem. 263:14621; Wilson, etal. (1992) J. Biol. Chem. 267:963-967; U.S. Pat. No. 5,166,320), directinjection of DNA uptake (see e.g., Acsadi, et al. (1991) Nature334:815-818; Wolff, et al. (1990) Science 247:1465-1468) or particlebombardment (see e.g., Cheng, et al. (1993) Proc. Natl. Acad. Sci. USA90:4455-4459; Zelenin, et al. (1993) FEBS Let. 315:29-32). Nucleic acidsequences encoding ATM kinase may be transferred into a fertilizedoocyte of a non-human animal to create a transgenic animal whichexpresses ATM kinase in one or more cell types. A transgenic animal isan animal having cells that contain a transgene, wherein the transgenewas introduced into the animal or an ancestor of the animal at aprenatal, e.g., an embryonic, stage. A transgene is a DNA which isintegrated into the genome of a cell from which a transgenic animaldevelops and which remains in the genome of the mature animal, therebydirecting the expression of an encoded gene product in one or more celltypes or tissues of the transgenic animal. Exemplary examples ofnon-human animals include, but are not limited to, mice, goats, sheep,pigs, cows or other domestic farm animals. Such transgenic animals areuseful, for example, for large-scale production of ATM kinase (genepharming) or for basic research investigations.

A transgenic animal may be created, for example, by introducing anucleic acid sequence encoding ATM kinase, typically linked toappropriate regulatory sequences, such as a constitutive ortissue-specific enhancer, into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, and allowing the oocyte to develop in apseudopregnant female foster animal. Intron sequences andpolyadenylation signals may also be included in the transgene toincrease the efficiency of expression of the transgene. Methods forgenerating transgenic animals, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866 and 4,870,009. A transgenic founder animal may beused to breed additional animals carrying the transgene. Transgenicanimals carrying a transgene encoding ATM kinase may further be bred toother transgenic animals carrying other transgenes, e.g., p53.

Once produced, the ATM kinase may be recovered from the culture mediumas a secreted polypeptide, although it also may be recovered from hostcell lysates when directly expressed without a secretory signal. WhenATM kinase is expressed in a recombinant cell other than one of humanorigin, the ATM kinase is completely free of proteins or polypeptides ofhuman origin. However, it is necessary to purify ATM kinase fromrecombinant cell proteins or polypeptides to obtain preparations thatare substantially homogeneous as to ATM kinase. As a first step, theculture medium or lysate is centrifuged to remove particulate celldebris. The membrane and soluble protein fractions are then separated.The ATM kinase may then be purified from the soluble protein fraction.ATM kinase thereafter is purified from contaminant soluble proteins andpolypeptides, as exemplified herein or with, for example, the. followingsuitable purification procedures: by fractionation on immunoaffinity orion-exchange columns; ethanol precipitation; reverse phase HPLC;chromatography on silica or on a cation-exchange resin such as DEAE;chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, Sephadex G-75; ligand affinitychromatography, and protein A Sepharose columns to remove contaminantssuch as IgG.

A further aspect of the invention provides a cell-based method ofidentifying agents which modulate activation of ATM kinase. Asindicated, ATM kinase in an unperturbed cell is in an inactive state.Upon DNA damage, ATM kinase is autophosphorylated at Ser¹⁹⁸¹ andconverted to an active state. Therefore, an agent which modulates thisautophosphorylation (i.e., activation) event may be identified in ascreening assay by contacting a cell producing full-length ATM kinase ora fragment thereof retaining ATM kinase activity with an agent anddetermining the phosphorylation state of Ser¹⁹⁸¹. Activation of ATMkinase may be modulated by blocking, inhibiting or decreasing activation(i.e., antagonizing) as well as activating, stimulating, or increasingactivation (i.e., agonizing). Enhancers of ATM kinase activation mayalso be identified in this screening assay as agents which increase therate or amount of ATM kinase activation following DNA damage. A typicalscreening assay for antagonists comprises contacting an unperturbed cellwith an agent, exposing said cell to a DNA damaging agent anddetermining whether said agent blocks or inhibits activation of ATMkinase. In contrast, a typical screening assay for agonists comprisescontacting an unperturbed cell with an agent and determining whethersaid agent stimulates activation of ATM kinase. Methods for identifyingthe activation state of ATM kinase in a cell are provided herein. In apreferred embodiment, activation of ATM kinase is identified bydetermining the phosphorylation state of Ser¹⁹⁸¹ using antibodies whichspecifically recognize the phosphorylation state of Ser¹⁹⁸¹ of ATMkinase.

A further aspect of the invention provides a cell-free method ofidentifying agents which modulate ATM kinase activity. Soluble ATMkinase protein produced by the method disclosed herein is isolated as amonomer in an active state with an unphosphorylated Ser¹⁹⁸¹ and uponaddition of a phosphate donor, autophosphorylates Ser¹⁹⁸¹ in the absenceof DNA/DNA damage. Therefore, a typical screening assay using solubleATM kinase protein comprises contacting soluble ATM kinase with anagent, exposing the soluble ATM kinase to a phosphate donor such as ATP,and detecting ATM kinase activity via autophosphorylation. The assay iscarried out under suitable assay conditions for autophosphorylation,such as those exemplified herein. The phosphate donor may be added withor after the agent. It is preferred that autophosphorylation is detectedusing an antibody which specifically recognizes the phosphorylationstate of Ser¹⁹⁸¹ of ATM kinase.

Agents which antagonize ATM kinase activity are useful asradiosensitizers or chemosensitizers in the treatment of a wide varietyof human tumors. Agents which agonize ATM kinase activity are useful asradioprotectors, cancer chemoprevention agents, and anti-aging agents.

It has now been found that cells treated with chloroquine prior toirradiation have increased cell survival by 30% (see Example 6).Interestingly, the cells treated, namely HeLa cells, are a knownradioresistant cell line in which additional radioprotection wasattained with chloroquine treatment.

Accordingly, the present invention provides a method for enhancing acellular response to DNA damage by administering an effective amount ofan agent which agonizes the activation of ATM kinase. It is contemplatedthat cellular responses are enhanced by an agonist to ATM kinase bypriming the cell to respond to agents which damage DNA including, butnot limited to, radiation, toxins, and carcinogens, and naturalprocesses or unnatural exposures to agents which cause oxidative DNAdamage. In this method, the agonist would be administered enough inadvance of exposure to the DNA damaging agent to provide the enhancingeffect. As used herein, an effective amount of ATM agonist is an amountwhich, for example, reduces DNA damage, reduces DNA mutation orincreases survival of cells exposed to a DNA damaging agent whencompared to cells exposed to the same DNA damaging agent and notreceiving an ATM agonist. Exemplary agonists which may be used inaccordance with the method of the present invention include chloroquineand Trichostatin A. In one preferred embodiment, the agonist to ATMkinase is radioprotectant for preventing damage to DNA caused by, forexample, ionizing or nuclear radiation. In another preferred embodiment,the agonist to ATM kinase is cancer chemopreventive for preventing tumorcell growth, progression and metastasis. Mice carrying supernumerarycopies of the p53 gene exhibit significant protection from cancer whencompared with normal mice (Garcia-Cao, et al. (2002) EMBO J.21:6225-35). Accordingly, a cancer chemoprevention agent may be used toactivate p53 via ATM kinase activation to enhance resistance to certaincancers. In another preferred embodiment, the agonist to ATM kinase isanti-aging. An agent which activates ATM kinase to enhance cellresponses to oxidative stress may slow the aging process.

As demonstrated herein, activation of ATM kinase induces phosphorylationand activation of p53. Manipulating p53-mediated pathways by way of ATMkinase activation is useful for enhancing cellular responses to avariety of agents which normally do not activate ATM kinase. Thus, inanother preferred embodiment of the present invention, an agent whichagonizes the activation of ATM kinase enhances cellular responses to DNAdamage via activation of p53-mediated pathways.

Agents which may be screened using the screening assays provided hereinencompass numerous chemical classes, though typically they are organicmolecules, preferably small organic compounds having a molecular weightof more than 100 and less than about 2,500 daltons. Agents comprisefunctional groups necessary for structural interaction with proteins,particularly hydrogen bonding, and typically include at least an amine,carbonyl, hydroxyl or carboxyl group, preferably at least two of thefunctional chemical groups. The agents often comprise cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups. Agents mayalso be found among biomolecules including peptides, antibodies,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof.

Agents are obtained from a wide variety of sources including librariesof synthetic or natural compounds. Alternatively, the antibodies of thisinvention may be used for the design and synthesis of either peptide ornon-peptide compounds (mimetics) specific to ATM kinase (see, e.g.,Saragovi, et al. (1991) Science 253:792-795).

A variety of other reagents may be included in the screening assays.These include reagents like salts, neutral proteins, e.g., albumin,detergents, etc. which may be used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Also,reagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, and thelike may be used. The mixture of components may be added in any order,that provides for the requisite binding.

Alternatively, the soluble ATM kinase provided herein may be used togenerate a crystal structure of ATM kinase. Once the three-dimensionalstructure of ATM kinase is determined, a potential agent (antagonist oragonist) can be examined through the use of computer modeling using adocking program such as GRAM, DOCK, or AUTODOCK (Dunbrack, et al. (1997)Folding & Design 2:27-42). This procedure can include computer fittingof potential agents to ATM kinase to ascertain how well the shape andthe chemical structure of the potential ligand will complement orinterfere with, the binding of ATM kinase domain with a substrate.Computer programs can also be employed to estimate the attraction,repulsion, and steric hindrance of the agent. Generally the tighter thefit (e.g., the lower the steric hindrance, and/or the greater theattractive force) the more potent the potential agent will be sincethese properties are consistent with a tighter binding constraint.Furthermore, the more specificity in the design of a potential agent themore likely that the agent will not interfere with related mammalianproteins. This will minimize potential side-effects due to unwantedinteractions with other proteins.

The invention is described in greater detail by the followingnon-limiting example.

EXAMPLE 1 Cell Culture, Immunofluorescence

293T cells, HeLa cells and 1070SK primary human foreskin fibroblasts(HFF) (>passage 20, ATCC) were cultured in Dulbecco's Modification ofEagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).GM00637 and GM09607 fibroblasts were grown in DMEM containing 15% FBS.GM00536 lymphoblast cells were grown in RPMI supplemented with 10% fetalcalf serum. 293T cells were transfected using FUGENE™ (Roche,Indianapolis, Ind.) and HeLa cells, GM00637 and GM09607 withLIPOFECTAMINE™ (INVITROGEN™ Corp., Carlsbad, Calif.). Metabolic labelingwas performed by pre-equilibrating cells in phosphate-free media forthree hours prior to the addition of 0.5 mCi/ml ³²P orthophosphate(PerkinElmer Life Sciences Inc., Boston, Mass.) for 30 minutes.Inhibition of DNA synthesis and analysis of G2/M checkpoint afterirradiation was assessed using well-known methods (e.g., Xu, et al.(2001) Mol. Cell. Biol. 21:3445-3450). Wortmannin was added to sampleson ice 15 minutes prior to reactions and 5 μM manganese and 1 μgsonicated calf thymus DNA were present or absent. Proteins werecrosslinked by incubating cells in formaldehyde/phosphate-bufferedsaline (PBS) for 10 minutes at room temperature. Formaldehyde was washedout using PBS containing 100 mM glycine and immunoprecipitation was thenperformed. Hypotonic swelling was performed for 1 hour in PBS containing0.45% glucose (w/v) and 1% FBS with the NaCl concentration reduced toeither 50 mM or 100 mM. HFF were incubated with chloroquine in DMEMcontaining 10% FBS for four hours. Prior to an eight hour incubationwith Trichostatin A, HFF was grown for 24 hours in DMEM containing 0.1%FBS. No cell death was observed in chloroquine or in hypotonicconditions and all cells recovered when put back in isotonic conditions.For immunofluoresence experiments, HFF grown on glass slides were fixedin 50% methanol/50% acetone for two hours at −20° C. Cells wereincubated with primary antibodies at 1/1000(H2AXγ; Upstate,Charlottesville, Va.) and secondary antibodies at 1/500(Cy3 anti-mouseand FITC anti-rabbit; Jackson ImmunoResearch Laboratories, Inc., WestGrove, Pa.) in PBS, 10% FBS for one hour.

EXAMPLE 2 Plasmids and Recombinant Protein Purification

FLAG®-tagged wild-type and kinase-inactive ATM are well-known in the art(e.g., Canman, et al., (1998) Science 281:1677-1679). Wild-type,FLAG®-tagged ATM was mutated using the QUIKCHANGE® site-directedmutagenesis kit (Stratagene, La Jolla, Calif.). The I-SceI expressionplasmid is well-known in the art (Rouet, et al. (1994) Proc. Natl. Acad.Sci. USA 91:6064-6068). GST fusion proteins were expressed in BL21 frompGEX-4T-1 (Amersham Pharmacia Biotech, Uppsala, Sweden). Fusion proteinswere purified and GST pull-down experiments performed by binding toglutathione-SEPHAROSE® beads (Sigma-Aldrich, St. Louis, Mo.) in PBS,0.5% NP-40, 1 mM AEBSF and 1 mM DTT. Bound proteins were washed fivetimes in the same buffer and eluted with 20 mM glutathione in 50 mMTris/HCl, pH 8.0. ATM kinase domain was expressed from pET28 (NOVAGEN®,Inc., Madison, Wis.). Recombinant ATM with N-terminal 6×-His and FLAG®tags was expressed from pYES2 (INVITROGEN™, Carlsbad, Calif.) in JEL1, aprotease-deficient S. cerevisiae strain that overexpresses thetranscription factor GAL4 driven by the GAL1 promoter (Lindsley and Wang(1993) J. Biol. Chem. 268:8096-8104). Following induction in 2%galactose for 16 hours yeast were lysed in 50 mM sodium phosphate pH8.0, 300 mM NaCl, 0.4 μM aprotinin, 1 mM AEBSF, 1× soy trypsin inhibitor(Roche, Indianapolis, Ind.), 1.5 mM pepstatin and 42 μM leupeptin bythree passages through a French press. Lysates were cleared bycentrifugation at 35,000 rpm in a 45Ti centrifuge (Beckman COULTER®,Inc., Fullerton, Calif.). For anti-FLAG® M2 sepharose (Sigma-Aldrich,St. Louis, Mo.) affinity purification TWEEN® 20 and NP-40 were added to1% and 0.5% respectively. FLAG® ATM was eluted in 100 μg/ml FLAG®peptide (Sigma-Aldrich, St. Louis, Mo.) in buffer A: 50 mM Tris (pH7.5), 150 mM NaCl, 1% TWEEN® 20, 0.5% NP-40, 50 mM NaF, 1 mM AEBSF, and1× protease inhibitor mixture (Roche, Indianapolis, Ind.).Size-exclusion chromatography was performed using a SUPERDEX® 200HR10/30 column (Amersham Pharmacia Biotech, Uppsala, Sweden) in 50 mMsodium phosphate, pH 8.0 containing 150 mM NaCl. For Ni-NTA affinitypurification, 20 mM imidazole was added to the lysates. 6×−His ATM waseluted from the nickel beads in 500 mM imidazole, pH 5.0, 300 mM NaCland 2 mM EDTA.

EXAMPLE 3 Immunoprecipitation, in vitro ATM Kinase Assays, and PeptideMapping

Mammalian cell extracts were prepared in buffer A. Cleared supernatantswere immunoprecipitated with anti-FLAG® M2 sepharose or anti-ATM D1611(Alligood, et al., (2000) Hybridoma 19:317-321) and protein A/G agarose.Beads were washed twice with buffer A and twice with RIPA buffer.Co-immunoprecipitation was performed in buffer B: 50 mM Tris (pH 7.5),150 mM NaCl, 0.5% TWEEN® 20, 0.2% NP-40, 50 mM NaF, 1 mM AEBSF, and 1×protease inhibitor mixture (Roche, Indianapolis, Ind.).

Ionizing irradiation induces phosphorylation of ATM at a single aminoacid residue. The residue and consequence of ATM phosphorylation wereexamined by transiently transfecting 293T cells with either FLAG®-taggedwild-type or kinase-inactive ATM and metabolically labeling with³²P-orthophosphate. The amount of radioactive orthophosphateincorporated into transfected wild-type ATM thirty minutes afterexposure of cells to 10 Gy IR. was approximately five-fold greater thanthat seen in unirradiated cells. In contrast, no such increase wasobserved following irradiation of cells that had been transfected withkinase-inactive ATM. Similarly, the amount of phosphate incorporatedinto endogenous ATM in immortalized lymphoblasts was markedly increasedfollowing IR. Short labeling periods were required for these experimentssince exposure of cells to these amounts of radioactive orthophosphatefor longer than thirty minutes damages DNA and obscures differencesbetween irradiated and unirradiated cells (Lim, et al. (2000) Nature404:613-617; Siliciano, et al. (1997) Genes Dev. 11:3471-3481).

Incorporation of radioactive phosphate into ATM also occurs in in vitroassays of ATM kinase activity (Canman, et al. (1998) Science281:1677-1679; Kim, et al. (1999) J. Biol. Chem. 274:37538-37543). Forin vitro kinase assays, beads were washed twice with buffer A, twicewith buffer A containing 0.5 M LiCl, and twice with kinase buffer: 20 mMHEPES (pH 7.5), 50 mM NaCl, 10 mM MgCl₂ and 10 mM MnCl₂. ATM kinasereactions were performed at 30° C. for five minutes in 50 μl of kinasebuffer containing 10 μCi of [γ-³²P] ATP and 1 μg of GST-fusionsubstrate. In vitro phosphorylation of ATM was not seen with thekinase-inactive ATM protein; was inhibited by exposure to 30 nM of thePI-3K inhibitor wortmannin; was dependent on the addition of thedivalent cation manganese; and was not dependent on addition ofexogenous DNA. Identical properties are characteristic of ATM in itsphosphorylation of target substrates: a concentration of 30 nMwortmannin effectively inhibits ATM kinase activity (Sarkaria, et al.(1998) Cancer Res. 58:4375-4382); its activity requires the presence ofmanganese (Canman, et al. (1998) Science 281:1677-1679; Kim, et al.(1999) J. Biol. Chem. 274:37538-37543); and exogenous DNA does notenhance its activity after immunoprecipitation from cells (Kim, et al.(1999) J. Biol. Chem. 274:37538-37543). Together with the observationthat ATM kinase activity was required for the increase in ATMphosphorylation seen during metabolic labeling of cells, these findingsindicate that ATM phosphorylates itself.

The site of phosphorylation in ATM was identified by radioactivelylabeling ATM protein, digesting with trypsin, and analyzing trypticphosphopeptides by sequential two-dimensional (2-D) electrophoresis andchromatography. Tryptic digestion of FLAG® ATM immobilized onnitrocellulose, 2-D resolution (electrophoresis and chromatography),manual Edman degradation and V8 digestion of peptides isolated fromthin-layer chromatography plates were performed using well-known methods(Meisenhelder et al. Current Protocols in Molecular Biology. Ausubel, F.M. et al. (eds.) (1999)). A single de novo phosphorylated peptide wasidentified in both transfected FLAG®-tagged ATM and in endogenous ATMisolated from irradiated cells. Since the increase in phosphorylation ofthis peptide was seen in vivo with wild-type, but not kinase-inactiveATM, this event is consistent with IR-inducible ATM autophosphorylation.

The ATM amino acid residue phosphorylated following IR was determined.The phosphopeptide identified by thin layer chromatography was isolatedand subjected to acid hydrolysis. Resolution of the labeled phosphoaminoacid by 2-D electrophoresis with unlabeled phosphoamino acid markersrevealed phosphoserine. There are 121 predicted tryptic peptides in ATMcontaining a single phosphoserine residue and many additional peptidescontaining two or more. Direct identification of the phosphopeptide byautomated sequencing and mass spectrometry failed, at least in part dueto difficulties in obtaining sufficient quantities of material.

The predicted migrations of tryptic serine-containing phosphopeptides inATM eliminated many candidates. Forty-one tryptic serinephosphopeptides, whose theoretical migration satisfied criteria for thatof the principal peptide at pH 1.9 and pH 4.72, were synthesized andsubjected to 2-D electrophoresis and chromatography along with trypticdigests of radioactively labeled ATM. The theoretical and observedmigrations of the 41 peptides were coincident at both pH's but thisnonetheless failed to identify the radiolabeled phosphopeptide.

Many kinases are activated by phosphorylation within their kinasedomains (Johnson and Noble (1996) Cell 85:149-158), therefore, eachserine in the kinase domain, as well as several other Ser-Gln sequencesin ATM, were subjected to site-directed mutagenesis. Individual serinesin wild-type, FLAG®-tagged ATM were mutated to glycine or alanine andthe mutants were expressed, immunoprecipitated and allowed toautophosphorylate in vitro. The labeled, mutant ATM proteins were thendigested with trypsin and the resulting tryptic phosphopeptides wereresolved by 2-D mapping. Surprisingly, the major phosphopeptide wasstill detected on 2-D maps of each ATM mutant, all of which retainedkinase activity. Therefore, none of the serines in the kinase domain ofATM are required for its kinase activity.

The major ATM phosphopeptide was identified. by use of secondaryproteolytic and chemical cleavage of the primary phosphopeptide. Afterin vivo radioactive labeling of ATM protein and purification of thephosphopeptide from the thin layer chromatography plate, the purifiedtryptic phosphopeptide was digested with V8 protease. The appearance ofa peptide with a new mobility on 2-D maps dictated the presence of aglutamic acid in the peptide. Furthermore, an unchanged chromatographicmobility after cyanogen bromide treatment of the purified trypticphosphopeptide in formic acid indicated that it did not containmethionine.

Manual Edman degradation was then performed on both the tryptic serinephosphopeptide and the derived V8/tryptic phosphopeptide. Resolution ofthe Edman degradation products revealed a secondary spot in each cyclesuggesting a derivatized peptide. If the peptide being treated in theEdman reaction contains a C-terminal lysine, the phenylisothiocyanatetreatment followed by acid treatment causes a derivatization of thelysine ε-amino group (Meisenhelder et al. Current Protocols in MolecularBiology. Ausubel, F. M. et al. (eds.) (1999)). Thus, the manual Edmanreactions indicated that the phosphopeptide of interest had a C-terminallysine. The second cycle of Edman degradation resulted in the generationof free phosphate from the V8/tryptic phosphopeptide. Thus, thephosphorylated serine is two amino acid residues to the C-terminal sideof a glutamic acid residue. Further cycles of Edman degradation wererequired to release the phosphate from the original trypticphosphopeptide, with generation of some free phosphate at cycle eightand substantial release at cycle nine. Thus, the phosphorylated serinein the larger peptide was eight or nine amino acid residues to theC-terminal side of either a lysine or arginine residue.

Although inefficiencies in the manual Edman degradation process causedifficulties when more than five or six cycles are required for aminoacid release (Meisenhelder et al. Current Protocols in MolecularBiology. Ausubel, F. M. et al. (eds.) (1999)), a partial sequence wasobtained for the major serine phosphopeptide ofXaa₅₋₆-Glu-Xaa-Ser-Xaa_(n)-Lys (SEQ ID NO:3) (where Xaa is any aminoacid other than Met, the N-terminus contains 5-6 residues, and thesequence preceding the C-terminal Lys is of unknown length, n). The onlytryptic peptide in ATM which meets these sequence requirements is the 19residue peptide1974-Ser-Leu-Ala-Phe-Glu-Glu-Gly-Ser-Gln-Ser-Thr-Thr-Ile-Ser-Ser-Leu-Ser-Glu-Lys-1992(SEQ ID NO:4). In addition, since V8 protease does not cleave at everyglutamic acid in a peptide sequence, the adjacent residues Glu¹⁹⁷⁸ andGlu¹⁹⁷⁹ explain the release of free phosphate at both cycles two andthree in the manual Edman degradation of the V8/tryptic phosphopeptide.The V8 phosphopeptide was likely a mixture of two peptides that wereunresolved on the 2-D map at pH 1.9, one of which had been cleaved atGlu¹⁹⁷⁸ and one at Glu¹⁹⁷⁹. It is noted that V8 did not digest thetryptic phosphopeptide at Glu¹⁹⁹¹ since the derivatization of Lys¹⁹⁹² inthe manual Edman degradation of the V8/tryptic phosphopeptide was seen.Ser¹⁹⁸¹ in ATM is conserved in mouse and Xenopus ATM, but is not foundin suspected homologues of less complex metazoans and is located in theN-terminus of the FAT domain, a region of approximately 500 amino acidresidues with some conservation across the PI-3K family of kinasesincluding Frap, ATM and Trapp (Bosotti, et al. (2000) Trends Biochem.Sci. 25:225-227). It is also noted that this is an ‘Ser-Gln’ site, thusindicating either an autophosphorylation event or phosphorylation by anATM family member.

EXAMPLE 4 Western Blotting and Antisera Production

Western blotting was performed with anti-FLAG® M5 (Sigma-Aldrich, St.Louis, Mo.), anti-ATM MAT3, anti-GST (Amersham Pharmacia Biotech,Uppsala, Sweden) or anti-6×-His (Sigma-Aldrich, St. Louis, Mo.).Anti-Ser¹⁹⁸¹ and anti-Ser¹⁹⁸¹-P specific antibodies were generated byimmunizing rabbits with KLH-conjugated synthetic peptidesSer-Leu-Ala-Phe-Glu-Glu-Gly-Ser-Gln-Ser-Thr-Thr-Ile-Ser-Ser (SEQ IDNO:5) (three animals) andSer-Leu-Ala-Phe-Glu-Glu-Gly-Ser(P)-Gln-Ser-Thr-Thr-Ile-Ser-Ser (SEQ IDNO:6) (6 animals), respectively (Rockland Immunochemicals,Gilbertsville, Pa.).

EXAMPLE 5 Autophosphorylation

Autophosphorylation of Ser¹⁹⁸¹ was determined usingglutathione-S-transferase (GST) fusion proteins containing variouslengths of ATM as substrates of the ATM kinase in in vitro kinasereactions. A polypeptide containing the appropriate serine residue at1981 was an excellent in vitro substrate, whereas a Ser¹⁹⁸¹→Ala mutantwas not phosphorylated by ATM whether the read-out was incorporation ofradioactive phosphate or binding by the α-Ser¹⁹⁸¹-P antibody. Inaddition, wortmannin concentrations of 20 μM or more effectivelyinhibited phosphorylation of Ser¹⁹⁸¹ following irradiation of humandiploid fibroblasts. This concentration of wortmannin inhibits both ATMand DNA-dependent protein kinase (DNA-PK), but not ataxia- andRad3-related protein (ATR), in vivo (Sarkaria, et al. (1998) Cancer Res.58:4375-4382). To exclude DNA-PK as a responsible kinase,phosphorylation of Ser¹⁹⁸¹ was determined in a cell line lacking DNA-PKactivity. The kinetics and levels of IR-induced phosphorylation ofSer¹⁹⁸¹ were identical to those in a similar cell line containing DNA-PKactivity.

In vivo experiments demonstrated that optimal phosphorylation of ATM wasdependent on the presence of active ATM kinase, however, the cells usedcontained endogenous wild-type ATM. Thus, even the kinase-inactivemutant of ATM was phosphorylated to some extent in these cells. Theimportance of ATM kinase activity in the phosphorylation of Ser¹⁹⁸¹ wasfurther analyzed using constructs encoding wild-type, kinase-inactive,and Ser¹⁹⁸¹→Ala ATM transfected into A-T cells such that the onlypotential source of ATM kinase activity was the transgene beingutilized. All of these constructs were expressed at similar levels inA-T cells, but only wild-type ATM was recognized by α-Ser¹⁹⁸¹-P, and itsbinding was increased several-fold within 30 minutes after exposure to10 Gy IR. Although some phosphorylation of kinase-inactive ATM wasobserved following transfection into 293T cells, which containendogenous ATM activity, transfection into a cell line that lacked ATMkinase activity showed no detectable phosphorylation on Ser¹⁹⁸¹.Therefore, phosphorylation of Ser¹⁹⁸¹ depends on the activity of the ATMkinase itself and the phosphorylation of transfected kinase-inactive ATMmust occur in trans.

Mutating Ser¹⁹⁸¹ did not abrogate ATM kinase activity in vitro, butconferred dominant inhibitory activity in cells. Expression vectorsencoding wild-type and Ser¹⁹⁸¹→Ala ATM proteins were transfected intoA-T fibroblasts and kinase activity was assessed by in vitro kinaseassays performed with the immunoprecipitated ATM proteins. Bothwild-type and Ser¹⁹⁸¹→Ala mutant ATM exhibited in vitro kinase activitydirected against a peptide containing the Ser¹⁵ target sequence in p53.To explore the in vivo activity of the Ser¹⁹⁸¹→Ala mutant, nucleic acidsequences encoding ATM kinase were introduced into HeLa cells and theintegrity of their ATM-dependent, IR-induced G2 and S-phase checkpointswas examined. Despite its kinase activity in the in vitro assay,Ser¹⁹⁸¹→Ala ATM mimicked kinase-inactive ATM in inhibiting both theIR-induced G2 checkpoint and S-phase replication arrest. Consistent withits abrogation of the IR-induced cell cycle checkpoints, the transfectedSer¹⁹⁸¹→Ala mutant also blocked the IR-induced phosphorylation ofendogenous ATM protein on Ser¹⁹⁸¹. Thus, whereas phosphorylation of ATMon Ser¹⁹⁸¹ does not appear to be required for kinase activity in vitro,expression of Ser¹⁹⁸¹→Ala ATM effectively inhibits the cellularactivities of endogenous ATM in a dominant-inhibitory manner.

EXAMPLE 6 Radioprotection Assay

HeLa cells were treated with 2 μg/ml of chloroquine for one hour, washedfor one hour, and irradiated at 2 or 6 Gy. Subsequently, 1000 cells wereplated and assessed for colony formation. Table 1 shows that exposure tochloroquine prior to irradiation increased cell survival by 30%. TABLE 1Average Number of Treatment Colonies* Std. Dev. 2 Gy 444 19.5Chloroquine + 2Gy 580 21.2 6 Gy 94.6 10.6 Chloroquine + 6 Gy 129 8.6*Averages were from five individual samples.

1-4. (canceled)
 5. A method of enhancing a cellular response to DNAdamage comprising administering an effective amount of an agent whichagonizes the activation of Ataxia-Telangiectasia Mutated kinase so thata cellular response to DNA damage is enhanced.
 6. The method of claim 5,wherein the agent is selected from the group comprising chloroquine orTrichostatin A.
 7. The method of claim 5, wherein the agent isradioprotectant.
 8. The method of claim 5, wherein the agent is cancerchemopreventive.
 9. The method of claim 5, wherein the agent isanti-aging.
 10. The method of claim 5, wherein the cellular response isvia p53 activation.
 11. (canceled)